In the search for new therapeutics, the pharmaceutical industry has increasingly turned to the techniques of combinatorial chemistry, parallel synthesis, and high throughput screening to generate and optimize lead compounds (Combinatorial Chemistry and Molecular Diversity in Drug Discovery Gordon and Kerwin, Eds., John Wiley & Sons, New York, 1998; The Combinatorial Index Bunin, Academic Press, New York, 1998; A Practical Guide to Combinatorial Chemistry Czarnik and DeWitt, Eds., American Chemical Society, Washington, D.C., 1997; High Throughput Screening: The Discovery of Bioactive Substances Devlin, Marcel Dekker, New York, 1997). These techniques can produce libraries of hundreds to hundreds of thousands--or more--of compounds in a short period of time. The libraries are then assayed against targets of interest, often in a highly automated fashion, to identify biologically active compounds. Libraries, which are simply collections of compounds, may be tightly focused around a specific template or contain a variety of unrelated templates. In many instances, the diversity of the library is an important design parameter.
On a basic level, the number of points of diversity on a molecular template or scaffold, i.e., the number of positions at which variation in structure may be introduced, has a practical effect on the ease with which large libraries may be created. When combinatorial techniques are employed, a template that contains three points of diversity would give rise to 8000 compounds if 20 components are used to derivatize each point and a total of 60 reactions are carried out (20.sup.3). However, a template with four points of diversity will yield over 50,000 compounds when 15 components are used at each point in a total of 60 reactions (15.sup.4). In general, large libraries may be created more efficiently on templates allowing more possibilities for derivatization.
In order to increase the chances of finding a biologically active compound for a particular target, it is usually desirable to synthesize a library spanning a range of both conformational space and chemical properties such as hydrophobicity and hydrogen bonding ability. At the same time, low molecular weight is often a goal as well, since compounds of less than 500 Daltons are perceived as more likely to have favorable pharmacokinetic properties in relation to higher molecular weight compounds. All these characteristics point to the continuing need for small compact templates that support a wide range of substituents and which are simple to synthesize.
Reverse-turns comprise one of three classes of protein secondary structure and display three (gamma-turn), four (beta-turns), or more (loops) amino acid side chains in a fixed spatial relationship to each other. Turns have proven important in molecular recognition events (Rose et al., Advances in Protein Chemistry 37:1-109, 1985) and have engendered a burgeoning field of research into small molecule mimetics of them (e.g., Hanessian et al., Tetrahedron 53:12789-12854, 1997). Many mimetics have either been external turn-mimetics which do not allow for the display of all the physiologically relevant side-chains (e.g., Freidinger et al., Science 210:656-8, 1980) or small, conformationally mobile cyclic peptide derivatives (e.g., Viles et al., Eur. J Biochem. 242:352-62, 1996). However, non-peptide compounds have been developed which closely mimic the secondary structure of reverse-turns found in biologically active proteins or peptides. For example, U.S. Pat. Nos. 5,475,085, 5,670,155 and 5,672,681 to Kahn and published PCT WO94/03494 to Kahn all disclose conformationally constrained, non-peptidic compounds which mimic the three-dimensional structure of reverse-turns. More recently, U.S. Pat. No. 5,929,237 to Kahn, and published PCT WO97/15577 to Kahn and PCT WO98/49168 to Kahn et al. disclosed additional, highly constrained bicyclic heterocycles as reverse-turn mimetics. Nevertheless, as no one template can mimic every type of turn, there remains a need in the art for additional reverse-turn templates.
Cell adhesion is critical to the viability of living organisms. Adhesion holds multicellular tissues together and directs embryonic development. It plays important roles in wound healing, eradication of infection and blood coagulation. Integrins are a family of cell surface proteins intimately involved in all of these functions. They have been found in nearly every type of human cell except red blood cells. Abnormalities in integrin function contribute to a variety of disorders including inflammatory diseases, heart attack, stroke, and cancer.
Integrins consist of heterodimers of .alpha. and .beta. subunits, non-covalently bound to each other. These cell surface receptors extend through the cell membrane into the cytoplasm. At least 15 different .alpha. and 9 different .beta. subunits are known. However, because most .alpha. proteins associate with only a single .beta. there are about 21 known integrin receptors. On the cell surface the heads of the two subunits contact each other to form a binding surface for extracellular protein ligands, allowing attachment to other cells or to the extracellular matrix. The affinity of these receptors may be regulated by signals from outside or within the cell. For example, recruitment of leukocytes to the site of injury or infection involves a series of adhesive interactions. Weak interaction between endothelial and leukocyte selectins and carbohydrates mediate transient adhesion and rolling of the leukocyte along the vessel wall. Various chemokines and other trigger factors released by the site of inflammation serve as signals to activate integrins from a quiescent to a high affinity state. These activated integrins then bind their cognate ligands on the surface of the endothelial cells, resulting in strong adhesion and flattening of the leukocyte. Subsequently the leukocyte migrates through the endothelium into the tissue below.
Integrin .alpha..sub.4.beta..sub.1 mediates cell adhesion primarily through binding to either vascular cell adhesion molecule-1 (VCAM-1) or an alternatively spliced variant of fibronectin containing the type III connecting segment (IIICS). A variety of cells involved in inflammation express .alpha..sub.4.beta..sub.1, including lymphocytes, monocytes, basophils and eosinophils, but not neutrophils. Monoclonal antibodies to the .alpha..sub.4 subunit have been used to validate .alpha..sub.4 -containing integrins as potential therapeutic targets in animal models of rheumatoid arthritis (Barbadillo et al., Springer Semin Immunopathol. 16:427-36, 1995; Issekutz et al., Immunology 88:569-76, 1996), acute colitis (Podolsky et al., J Clin. Invest. 92:372-80, 1993), multiple sclerosis (Yednock et al., Nature 356:63-6, 1992), asthma (Abraham et al., J Clin. Invest. 93:776-87, 1994) U.S. Pat. No. 5,871,734) and diabetes (Tsukamoto et al., Cell Immunol. 165:193-201, 1995). More recently, low molecular weight peptidyl derivatives have been produced as competitive inhibitors of .alpha..sub.4.beta..sub.1 and one has been shown to inhibit allergic airway responses in sheep (Lin et al., J Med. Chem. 42:920-34, 1999).
It has been shown that a key sequence in IIICS involved in binding to .alpha..sub.4.beta..sub.1 is the 25 residue peptide CS1, and within that sequence the minimally recognized motif is the tripeptide, LDV. A similar sequence, IDS, has been implicated in the binding of VCAM-1 to .alpha..sub.4.beta..sub.1. X-ray crystal structures of an N-terminal two-domain fragment of VCAM-1 show that the IDS sequence is part of an exposed loop linking two beta-strands (Jones et al., Nature 373:539-44, 1995; Wang et al., Proc. Natl. Acad. Sci. USA 92:5714-8, 1995). Cyclic peptides and derivatives thereof which adopt reverse-turn conformations have proven to be inhibitors of VCAM-1 binding to .alpha..sub.4.beta..sub.1 (WO 96/00581; WO 96/06108; Doyle et al., Int. J Pept. Protein Res. 47:427-36, 1996). In addition, a number of potent and selective (versus .alpha..sub.4.beta..sub.1 ) cyclic peptide-based inhibitors have been discovered (Jackson et al., J Med. Chem. 40:3359-68, 1997). Several non-peptidyl beta-turn mimetics have also been reported to bind .alpha..sub.4.beta..sub.1 with IC.sub.50 s in the low micromolar range (Souers et al., Bioorg. Med. Chem. Lett. 8:2297-302, 1998). Numerous phenylalanine and tyrosine derivatives have also been disclosed as inhibitors of .alpha..sub.4.beta..sub.1 (WO 99/06390; WO 99/06431; WO 99/06433; WO 99/06434; WO 99/06435; WO 99/06436; WO 99/06437; WO 98/54207; WO 99/10312; WO 99/10313; WO 98/53814; WO 98/53817; WO 98/58902). However, no potent and orally available small molecule inhibitors have been disclosed.
A related integrin, .alpha..sub.4.beta..sub.7, is expressed on the surface of lymphocytes and binds VCAM-1, fibronectin and mucosal addressin cell adhesion molecule 1 (MAdCAM-1). Integrin .alpha..sub.4.beta..sub.7 and MAdCAM mediate recirculation of a subset of lymphocytes between the blood, gut, and lymphoid tissue. Similar to VCAM-1 and Fibronectin CS-1 there is a tripeptide sequence, LDT, present on the CD loop of MAdCAM-1 which is important for recognition by .alpha..sub.4.beta..sub.7. An X-ray crystal structure shows this sequence is also part of a turn structure(Tan et al., Structure 6:793-801, 1998). Recent studies have shown that .alpha..sub.4.beta..sub.7 may also play a part in diseases such as asthma (Lobb et al., Ann. NYA cad. Sci. 796:113-23, 1996), inflammatory bowel disease (Fong et al., Immunol. Res. 16:299-311, 1997), and diabetes (Yang et al., Diabetes 46:1542-7, 1997). In addition, while .alpha..sub.4 integrins appear to be down-regulated in carcinomas such as cervical and prostate, they appear to be up-regulated in metastatic melanoma (Sanders et al., Cancer Invest. 16:329-44, 1998), suggesting that inhibitors of .alpha..sub.4.beta..sub.1 and .alpha..sub.4.beta..sub.7 may be useful as anticancer agents.
While significant advances have been made in the synthesis and identification of conformationally constrained, reverse-turn mimetics, there is still a need in the art for small molecules that mimic the secondary structure of peptides. There is also a need in the art for libraries containing such members, particularly those small templates capable of supporting a high diversity of substituents. In addition, there is a need in the art for techniques for synthesizing these libraries and screening the library members against biological targets to identify bioactive library members. Further, there is a need in the art for small, orally available inhibitors of integrins, for use in treating inflammatory diseases and cardiovascular diseases, as well as some cancers. In particular there is a need for inhibitors of .alpha..sub.4.beta..sub.1 and .alpha..sub.4.beta..sub.7, for use in the treatment of rheumatoid arthritis, asthma, diabetes and inflammatory bowel disease.
The present invention fulfills these needs, and provides further related advantages.